Diabetes is a chronic disorder of glucose metabolism and a major cause of premature mortality. The potential use of replacement beta cells as therapy for diabetes requires an ability to culture such cells while maintaining their functional status. Glucose stimulated insulin secretion (GSIS) is lost in long-term cultured MIN6 heterogeneous cells. MIN6 B1, a clonal sub-line derived from MIN6, has been described as highly glucose-responsive. This study aimed to investigate the GSIS function, changes in gene expression and, subsequently, to develop possible experimental approaches to overcome this loss. Understanding the molecular basis for loss of GSIS may contribute to better culture conditions for islets in transplantation programmes; it may also add to our understanding of beta cell insensitivity to high blood glucose in Type 2 diabetes. Whole genome microarray analysis on biological triplicate samples of glucoseresponsive low passage (p19) (MIN6 B1(GSIS)) and non glucose-responsive high passage (p23) (MIN6 B1(Non-GSIS)) was performed. 111 differentially-regulated genes were identified and 16 gene transcripts were selected based on p-value <0.05; fold change 1.2; difference 100 and validated using qRT-PCR including Txnip, Gcg and Pcsk9. Txnip was up-regulated whereas Gcg and Pcsk9 were down-regulated in MIN6 B1(Non-GSIS) compared to MIN6 B1(GSIS). siRNA and shRNA silencing of Txnip in MIN6(H) (non-responsive) significantly increased the GSIS. Over-expression of Txnip cDNA in MIN6(L) (glucose-responsive) caused significant loss of GSIS. siRNA silencing of Gcg and Pcsk9 caused a significant loss of GSIS in MIN6(L) compared to scrambled-transfected cells. Over-expression of Gcg cDNA in MIN6(H) increased GSIS. In parallel, in an attempt to identify more reliable biomarkers for diabetes, we also investigated if extracellular mRNAs are reproducibly detectable in conditioned medium (CM) from a range of insulin-producing cell types and in serum specimens from Type 2 diabetes and controls. Pdx1, Npy, Egr1, Pld1, Chgb, Ins1, Ins2, and betaactin from MIN6(L), MIN6(H), and MIN6 B1 cells and their CM suggests that beta cells transcribe and release these mRNAs into their culture environment.This study was subsequently translated to analysis of serum from people with Type 2 diabetes and controls to help establish the clinical relevance of these findings. The result from this clinical phase of the study indicated Txnip to be up-regulated and Egr1 to be down-regulated in Type 2 diabetes compared to controls.